The aim of this proposal is to examine the cellular interactions underlying changes in the developmental expression of gamma aminobutyric acid/benzodiazepine (GABA-A/BZ) receptor subunit mRNAs and ligand binding sites in the murine inferior olivary nucleus. The GABA-A/BZ receptor is thought to be a pentameric chloride channel comprised of selected alpha, beta, gamma, delta and rho subunits, the majority of which have several isoforms: alpha 1-6, beta 1-4, gamma 1-4 and rho 1-2. We have shown that, of the thirteen subunit variants present in the mammalian central nervous system, the alpha 2-5, beta 3, gamma 1-2 are expressed at significant levels in the inferior olivary nucleus. Two clearly different temporal patterns of GABA-A/BZ receptor subunit mRNA expression were observed: The expression of alpha3, alpha5, beta3 and gamma2 mRNAs was at a peak during perinatal development, followed by rapid downregulation thereafter. Conversely, alpha2, alpha4 and gamma1 mRNA expression was very low, or absent, during early development, and a pronounced increase was observed at the end of the first postnatal weeks Inferior olivary neurons are the primary source of climbing fibers innervating the Purkinje cells of the cerebellar cortex. The timecourse of afferent and efferent olivary connections precisely parallels the GABA-A/BZ receptor subunit changes in olivary neurons: During postnatal week two, olivary climbing fiber innervation of the target Purkinje cell regresses; the number of climbing fiber terminals innervating each Purkinje cell is reduced from greater than 3:1, as observed in neonates, to the 1:1 relationship observed in the adult cerebellar cortex. During the same period, olivary neurons become densely innervated by GABAergic afferents. This raises the possibility that the observed developmental switches in subunit expression may be related to epigenetic factors, such as a reduction in neurotrophic factors from the target Purkinje cell, or to stimulation by GABAergic cells of the deep cerebellar and other afferent nuclei. We will explore the role of a number of possible epigenetic mechanisms which may regulate GABA-A/BZ receptor subunit switching during development. These include the role of efferent and afferent connections, potential neurotrophic factors and increased electrical activity. In addition, the effect of subunit gene- deletion, and the generalizability of the subunit switching phenomenon will be studied.
